Electricity-producing genetically engineered bacteria used in microbial fuel cell, and construction method and application thereof
A technology of genetically engineered bacteria and fuel cells, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc.
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Embodiment 1
[0034] This example illustrates the construction of a target gene nad The intermediate plasmid pMD19-T- nad e. The process includes:
[0035] 1. Design and synthesize upstream and downstream primers,
[0036] Primer1 upstream primer: 5'-CGCTGTCTGGAGGGTTCAATGAC -3'
[0037] Primer2 downstream primer: 5'- CGCACAATCCAATATGTGC -3'.
[0038] 2. Escherichia coli E. coli DH5α genomic DNA was used as a template, and the target gene fragment was amplified by PCR. The reaction conditions were: pre-denaturation at 94 °C for 3 min; 30 cycles at 94 °C for 30 s, 52 °C for 30 s, 72 °C for 70 s, and 72 °C for 5 min. PCR products were detected by 0.8% agarose electrophoresis ( figure 2 ), recover the 828bp fragment, and purify the amplified by Genray kit nadAfter the E gene (Gene ID: 946946), perform T-A cloning according to the instruction manual of the purchased PMD19-T Simple plasmid, and the target gene nad E was connected to the sequencing carrier PMD19-T Simple plasmid, and...
Embodiment 2
[0040] This example illustrates the construction of an expression gene nad E recombinant plasmid pBBR1MCS- nad e. gene nad The recombinant plasmid pBBR1MCS- nad Build of E:
[0041] 1. Synthetic with Hind The upstream primer of the III restriction site and the Bam Downstream primers of the HI restriction site:
[0042] Primer3 upstream primer: 5'- CCC AAGCTT ATGACATTGCAACAAC -3' (the underlined part is Hind III restriction site);
[0043] Primer4 downstream primer: 5'-CGC GGATCC TTACTTTTTTCCAGAAATC -3 (the underlined part is Bam HI restriction site);
[0044] 2. Using pMD19-T- nad E is the template, and the target gene is amplified by PCR nad E (Gene ID: 946946), the reaction conditions are: 95°C, 10 min; (95°C for 30 s, 52°C for 30 s, 72°C for 70 s, 30 cycles); 72°C, 10 min. Purify the amplified enzyme cleavage site by the purification kit of Genray company nad After the E gene, it was connected with the pBBR1MCS-5 expression plasmid digested by th...
Embodiment 3
[0046] This embodiment illustrates the NAD of the whole cell protein solution of the electricity-producing genetically engineered bacteria constructed + Synthetic enzyme activity.
[0047] 1. Determination of protein concentration
[0048] Protein concentration was determined by the method of Bradford (1976).
[0049] 2. Preparation of NADH standard curve.
[0050] Dilute NADH into 8 different concentration gradients (0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.1mM) with 50mmol / L Tris-HCl buffer solution, measure the absorbance value at 340nm respectively, and then use OD 340 The value is the ordinate, the NADH concentration is the abscissa, and the standard curve is drawn (see Figure 8 ).
[0051] 3.NAD + Synthetic Enzyme Activity Assay
[0052] 1) Strain activation: Streak the preserved bacterial solution of the electrogenic engineered bacterium PA-nadE on an LB plate containing 50 μg / mL gentamicin, and pick a single colony grown on the plate to 5 mL of LB medium (pH =7.0),...
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